Regulation of peroxisomal fatty acid transport in plants

ABSTRACT

A nucleic acid which encodes a peroxisomal fatty acid transporter, uses thereof and a method of genetic manipulation of peroxisomal fatty acid transport and/or Metabolism. The nucleic acid and its products are especially for use in regulation of peroxisomal fatty acid transport in plant and in controlling the spectrum of fatty acids which can be utilised by the plant.

[0001] The present invention relates to an isolated nucleic acid, usesthereof and a method of genetic manipulation of peroxisomal fatty acidtransport and/or metabolism, means therefor and products thereofespecially for use in regulation of plant growth and in controlling thespectrum of fatty acids which can be utilised by the plant.

BACKGROUND TO THE INVENTION

[0002] Fatty acids are major carbon and energy stores in the seeds ofmany agriculturally important species. For the plant they are essentialreserves that support germination, and seedling establishment until theplant can manufacture its own building blocks and energy throughphotosynthesis. Recent published data shows that blocking themobilisation of these fatty acids prevents or severely compromisesestablishment (Hayashi et al., 1998, Germain et al., 2001). Clearly,efficient germination and seedling establishment are of vital importanceto farmers. In addition, the fatty acids deposited in seeds renders themimportant foodstuffs for humans and animals. There is considerableinterest in modifying the levels and composition of fatty acids in seedsto improve their nutritional quality and health benefit. Furthermore,there is considerable interest in engineering crop plants to producenovel fatty acids with industrial benefit. These can be used asfeedstocks for the chemical and healthcare industries, with the aim ofreducing reliance on petrochemical feedstocks, it is therefore desirableto produce these molecules in a more ecologically friendly andsustainable way and to develop non-food crops for European agriculture.

[0003] Plants that are engineered to produce altered fatty acids rarelyproduce economically viable levels in seeds. The reasons for this areprobably complex but one factor may be their turnover, i.e. someproportion is broken down as they are made. Furthermore, if high levelscan be achieved this may compromise the ability of these plants togerminal and establish if these altered fatty acids cannot be usedefficiently. Clearly this is detrimental to the commercialisation ofthese plants.

[0004] Fatty acids in seeds are stored in oil bodies in the form oftriacylglycerols (3 fatty acid molecules joined to a glycerol backbone)which are laid down during seed development. During germination freefatty acids are released by the action of lipases and the fatty acidsenter the β-oxidation pathway which is housed within a specialisedorganelle the glyoxysome. The fatty acids are then metabolised toproduce energy and building blocks for the cell. Control of biochemicalpathways frequently resides near the beginning of the pathway and inseveral cases transport steps have been shown to exert high flux controlcoefficients. This means that transporting a molecule, for example, fromcompartment A to compartment B is often an important step in determiningthe overall rate of the pathway.

[0005] There is some evidence to suggest from human studies thatproteins of the ATP Binding Cassette family (referred to as ABCs) areinvolved in fatty acid transport ABCs are integral membrane proteinsthat transport a wide variety of molecules across membranes. It is knownfrom the prior art that X-linked adrenoleukodystrophy (X-ALD) isassociated with a particular gene mutation (Moser et al., 1993). Theclinical symptoms of the disease, results in increasing neurologicalimpairment, progressive mental and physical disability, and eventuallydeath in late childhood or early teens. Biochemically these patientsfail to break down very long chain fatty acids. The gene mutated inX-ALD is an ABC transporter closely related to but not identical toanother mammalian peroxisomal ABC transporter, PMP70. It is now knownthat there are 4 of these peroxisomal ABC genes in humans (PMP70,PMP70R, ALD and ALDR. In addition, two homologous genes have beenidentified in yeast S. cerevisiae (PXA1 and PXA2 also known as PAT1 andPAT2) and have been shown to be transporters of fatty acyl CoAs (Hettemaet al., 1996). PXA2 is also known as the COMATOSE (CTS) gene (Russell etal., 2000; Footitt et al., 2002).

[0006] Glyoxysomes are cytoplasmic organelles unique to plants.Glyoxysomes are a specialised form of peroxisomes but they also containenzymes of the glyoxalate cycle. They are abundantly present in theendosperm or cotyledons of oil-rich seeds. It is not known how fattyacids are transported into glyoxysomes.

[0007] It is therefore desirous to identify a transport protein or aregulator protein that is involved with the rate of entry of fatty acidsinto the degradation pathway; Identification and characterisation of theproteins that transport fatty acids into glyoxysomes would offer anattractive target for biotechnology with a view to repress or promotegrowth or to alter the spectrum of fatty acids which can be utilised bythe plant

STATEMENTS OF THE INVENTION

[0008] According to a first aspect of the invention there is provided anisolated nucleic acid comprising a nucleotide sequence which encodes apolypeptide which functions as a fatty acid transporter in plantsselected from the group consisting of:

[0009] (i) a nucleic acid sequence depicted in SEQ ID NO: 1,

[0010] (ii) a nucleic acid sequence which is derived from the sequencedepicted in SEQ ID NO: 1 according to the degeneracy of the geneticcode,

[0011] (iii) derivatives of the sequence depicted in SEQ ID NO: 1, whichencodes polypeptides having preferably at least 30% homology to thesequence encoding amino acid sequences depicted in SEQ ID NO: 2 andwhich sequences function as a fatty acid transporter.

[0012] The nucleic acids of the present invention are convenientlyreferred to as the CTS gene.

[0013] Throughout this specification and the claims which follow, unlessthe context requires otherwise, the word “comprise”, or variations suchas “comprises” or “comprising”, will be understood to imply theinclusion of a stated integer or group of integers but not the exclusionof any other integer or group of integers. Accordingly, one aspect ofthe invention pertains to isolated nucleic acid molecules (e.g., cDNAs)comprising a nucleotide sequence encoding a fatty acid transporterprotein or biologically active portions thereof, as well as nucleic acidfragments suitable as primers or hybridization probes for the detectionor amplification of fatty acid transporter—encoding nucleic acid (e.g.,DNA or mRNA). In particularly preferred embodiments, the isolatednucleic acid molecule comprises one of the nucleotide sequences setforth in Sequence SEQ ID NO: 1 or the coding region or a complementthereof of one of these nucleotide sequences. In other particularlypreferred embodiments, the isolated nucleic acid molecule of theinvention comprises a nucleotide sequence which hybridizes to or is atleast about 50%, preferably at least about 60%, more preferably at leastabout 70%, 80% or 90%, and even more preferably at least about 95%, 96%,97%, 98%, 99% or more homologous to a nucleotide sequence as in SequenceSEQ ID NO: 1, or a portion thereof. In other preferred embodiments, theisolated nucleic acid molecule encodes one of the amino acid sequencesset forth in Sequence SEQ ID NO: 2. The preferred fatty acidtransporter-gene of the present invention also preferably possess atleast one of the fatty acid transporter activities described herein.These may include transport of fatty acids and/or acyl CoAs of varyingchain lengths, degree of unsaturation and substitution, and theiranalogues and derivatives and/or other amphipathic molecules such as 2,4dichlorophenoxybutyric acid and indole butric acid and their analoguesand derivatives.

[0014] In another embodiment, the isolated nucleic acid molecule is atleast 15 nucleotides in length and hybridizes under stringent conditionsto a nucleic acid molecule comprising a nucleotide sequence of SEQ IDNO: 1; Preferably, the isolated nucleic acid molecule corresponds to anaturally-occurring nucleic acid molecule. More preferably, the isolatednucleic acid encodes a naturally-occurring Arabidopsis thaliana fattyacid transporter, or a biologically active portion thereof.

[0015] Alternatively, the isolated fatty acid transporter can comprisean amino acid sequence which is encoded by a nucleotide sequence whichhybridizes, e.g., hybridizes under stringent conditions, or is at leastabout 50%, preferably at least about 60%, more preferably at least about70%, 80%, or 90%, and even more preferably at least about 95%, 96%, 97%,98,%, or 99% or more homologous, to a nucleotide sequence of SEQ IDNO: 1. It is also preferred that the preferred forms of fatty acidtransporters also have one or more of the fatty acid transporteractivities described herein Nucleic acid molecules corresponding tonatural variants and non-Arabidopsis thaliana homologues, derivatives oranalogues of the Arabidopsis thaliana fatty acid transporter cDNA of theinvention can be isolated based on their homology to Arabidopsisthaliana fatty acid transporter nucleic acid disclosed herein using theArabidopsis thaliana cDNA, or a portion thereof, as a hybridizationprobe according to standard hybridization techniques under stringenthybridization conditions. Accordingly, in another embodiment, anisolated nucleic acid molecule of the invention is at least 15nucleotides in length and hybridizes under stringent conditions to thenucleic acid molecule comprising a nucleotide sequence of SEQ BD NO: 1.In other embodiments, the nucleic acid is at least 25, 50, 100, 250 ormore nucleotides in length. As used herein, the term “hybridizes understringent conditions” is intended to describe conditions forhybridization and washing under which nucleotide sequences at least 60%homologous to each other typically remain hybridized to each other.Preferably, the conditions are such that sequences at least about 65%,more preferably at least about 70%, and even more preferably at leastabout 75% or more homologous to each other typically remain hybridizedto each other. Such stringent conditions are known to those skilled inthe art and can be found in Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred, non-limitingexample of stringent hybridization conditions are hybridization in 6×sodium chloride/sodium citrate (═SSC) at about 45° C., 25, followed byone or more washes in 0.2×SSC, 0.1% SDS at 50-65° C. As known by theskilled artisan such hybridization conditions differ depending on thetype of the nucleic acid and if for example organic solvents are presentin view of the temperature and the concentration of the buffer. Thetemperature for example differs under “standard hybridizationconditions” depending on the type of the nucleic acid between 42° C. and58C in aqueous buffer with a concentration of 0.1 to 5×SSC (pH 7.2). Inthe event that organic solvent is present in the before mentioned bufferfor example 50% formamide the temperature under standard conditions isabout 42° C.

[0016] Preferably hybridisation conditions for DNA:DNA-hybrids are forexample 0.1×SSC and 20° C. to 45° C., preferably between 30° C. to 45-C.Preferably hybridisation conditions for DNA:RNA-hybrids are for example0.1×SSC and 30-C to 55-C, preferably between 45° C. to 55° C. The beforementioned hybridization temperatures are estimated for example for anucleic acid of about 100 bp (=base pairs) in length with G+C-content of50% in the absence of formamide. The skilled worker knows how toestimate the necessary hybridization conditions according to textbookssuch as the one mentioned above or from the following textbooks Sambrooket al., “Molecular Cloning”, Cold Spring Harbor Laboratory, 1989; Hamesand Higgins (eds.), 1985, “nucleic Acids Hybridization: A PracticalApproach”, IRL Press at Oxford University Press, Oxford; Brown (ed),1991, “Essential Molecular Biology: A Practical Approach”, IRL Press atOxford University Press, Oxford. This is also true for stringent or lowstringent hybridization conditions.

[0017] Preferably, an isolated nucleic acid molecule of the inventionhybridizes under stringent conditions to a sequence of SEQ ID NO: 1corresponds to a naturally-occurring nucleic acid molecule. As usedherein, a “naturally-occurring” nucleic acid molecule refers to an RNAor DNA molecule having a nucleotide sequence that occurs in nature(e.g., encodes a natural protein); In one embodiment, the nucleic acidencodes a natural Arabidopsis thaliana fatty acid transporter.

[0018] In addition to naturally-occurring variants of the fatty acidtransporter sequence that may exist in the population, the skilledartisan will further appreciate that changes can be introduced bymutation into a nucleotide sequence of SEQ ID NO: 1, thereby leading tochanges in the amino acid sequence of the encoded fatty acidtransporter, without altering the functional ability of the fatty acidtransporter. For example, nucleotide substitutions leading to amino acidsubstitutions at “non-essential” amino acid residues can be made in asequence of SEQ ID NO: 1. A “non-essential” amino acid residue is aresidue that can be altered from the wild-type sequence of one of thefatty acid transporters (SEQ ID NO: 2) without altering the activity ofsaid fatty acid transporter, whereas an “essential” amino acid residueis required for fatty acid transporter activity. Other amino acidresidues, however, (e.g., those that are not conserved or onlysemi-conserved in the domain having fatty acid transporter activity) maynot be essential for activity and thus are likely to be amenable toalteration without altering fatty acid transporter activity.

[0019] In another embodiment, the isolated nucleic acid molecule encodesa protein or portion thereof wherein the protein or portion thereofincludes an amino acid sequence which is sufficiently homologous to anamino acid sequence of sequence SEQ ID NO: 2 such that the protein orportion thereof maintains a fatty acid transporter activity. Preferably,the protein or portion thereof encoded by the nucleic acid moleculemaintains the ability to participate in the metabolism of compoundsnecessary for the construction of fatty acids especially PUFAs orcellular membranes of plants or in the transport of molecules acrossthese membranes. In one embodiment, the protein encoded by the nucleicacid molecule is at least about 50%, preferably at least about 60%, andmore preferably at least about 70%, 80%, or 90%4 and most preferably atleast about 95%, 96%, 97%, 98%, or 99% or more homologous (=identity) toan amino acid sequence of Sequence SEQ ID NO: 2.

[0020] Further, DNAs of which code for proteins of the presentinvention, or DNAs which hybridize to that of SEQ ID NO:1 but whichdiffer in codon sequence from SEQ ED NO: 1 due to the degeneracy of thegenetic code, are also part of this invention. The degeneracy of thegenetic code, which allows different nucleic acid sequences to code forthe same protein or peptide, is well known in the literature. See, e.g.,U.S. Pat. No. 4,757,006 to Toole et al. at Col. 2, Table 1.

[0021] Sequence identity: the similarity between two nucleic acidsequences, or two amino acid sequences, is expressed in terms of thesimilarity between the sequences, otherwise referred to as a sequenceidentity. Sequence identity is frequently measured in terms ofpercentage identity (or similarity or homology); the higher thepercentage, the more similar the two sequences are. Homologues ororthologues of the protein, and the corresponding cDNA or gene sequence,will possess a relatively high degree of sequence identity when alignedusing standard methods. This homology will be more significant when theorthologous proteins or genes or cDNAs are derived from species that aremore closely related (e.g., human and chimpanzee sequences), compared tospecies more distantly related (e.g. human and C. elegans sequences).

[0022] Methods of alignment of sequences for comparison are well knownin the art. Various programs and alignment algorithms are described in:Smith & Waterman Adv. Appl. Math. 2: 482, 1981; Needleman & Wunsch J.Mol. Biol. 48: 443, 1970; Pearson & Lipman Proc. Natl. Acad. Sci. USA85: 2444, 1988; Higgins & Sharp Gene, 73: 237-244, 1988; Higgins & SharpCABIOS 5: 151-153, 1989; Corpet et al. Nuc. Acids Res. 16, 10881-90,1988; Huang et al. Computer Appls. In the Biosciences 8, 155-65, 1992;and Pearson et al. Meth Mol. Bio.24, 307-31, 1994. Altschul et al. (J.Mol. Biol. 215:403410, 1990), presents a detailed consideration ofsequence alignment methods and homology calculations.

[0023] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul etal: J. Mol. Biol. 215:403410, 1990) is available from several sources,including the National Center for Biotechnology Information (NCBI,Bethesda, Md.) and on the Internet, for use in connection with thesequence analysis blastp, blastn, blastx, tblastn and tblastx. By way ofexample, for comparisons of amino acid sequences of greater than about30 amino acids, the Blast 2 sequences function is employed using thedefault BLOSUM62 matrix set to default parameters, (gap existence costof 11, and a per residue gap cost of 1). When aligning short peptides(fewer than around 30 amino acids), the alignment may for example beperformed using the Blast 2 sequences function, employing the PAM30matrix set to default parameters (open gap 9, extension gap 1penalties).

[0024] According to a yet further aspect of the invention there isprovided a gene construct comprising an isolated nucleic acid having thesequence SEQ ID NO: 1 as herein before described, wherein the nucleicacid is functionally linked to one or more regulatory signals.

[0025] Accordingly, another embodiment of the invention is a novel geneconstruct comprising an isolated nucleic acid derived from a plant whichencodes a polypeptide which functions as fatty acid transporter or thegene sequence of SEQ ID No. 1, its homologous, derivatives or analogousas defined above which have been functionally linked to one or moreregulatory signals, advantageously to increase gene expression. Examplesof these regulatory sequences are sequences to which inducers orrepressors bind and thus regulate the expression of the nucleic acid. Inaddition to these novel regulatory sequences, the natural regulation ofthese sequences in front of the actual structural genes can still bepresent and, where appropriate, have been genetically modified so thatthe natural regulation has been switched off and the expression of thegenes has been increased. The gene construct can, however, also have asimpler structure, that is to say no additional regulatory signals havebeen inserted in front of the sequence SEQ ID No. 1 or its homologs, andthe natural promoter with its regulation has not been deleted. Instead,the natural regulatory sequence has been mutated so that regulation nolonger takes place, and gene expression is enhanced. The gene constructmay additionally advantageously comprise one or more so-called enhancersequences functionally linked to the promoter and making increasedexpression of the nucleic acid sequence possible. It is also possible toinsert at the 3′ end of the DNA sequences additional advantageoussequences, such as further regulatory elements or terminators. The fattyacid transporter genes may be present in one or more copies in the geneconstruct. It is advantageous for further genes to be present in thegene construct for insertion of further genes into organisms.

[0026] Advantageous regulatory sequences for the novel process arepresent, for example, in promoters such as cos-, tac-, trp-, tet-,trp-tet-, lpp-, lac-, lpp-lac-, lacIq-, T7-, T5-, T3-, gal-, trc-, ara-,SP6-, I-PR- or l-PL-promoter and are advantageously used inGram-negative bacteria Further advantageous regulatory sequences arepresent, for example, in the Gram-positive promoters amy and SPO2, inthe yeast or fungal promoters ADC1, MFa, AC, P-60, CYC1, GAPDH, TEF,rp28, ADH or in the plant promoters CaMV/35S [Franck et al., Cell 21(1980) 285-294], PRP1 [Ward et al., Plant. Mol. Biol. 22 (1993)], SSU,OCS, lib4, usp, STLS1, B33, nos or in the ubiquitin or phaseolinpromoter. Also advantageous in this connection are inducible promoterssuch as the promoters described in EP-A-0 388 186 (benzyl sulfonamideinducible), Plant J. 2, 1992: 397-404 (Gatz et al., Tetracyclininducible), EP-A-0 335 528 (abscisic acid inducible) or WO 93/21334(ethanol or cyclohexenol inducible). Additional useful plant promotersare the cytosolic FBPase promotor or ST-LSI promoter of the potato(Stockhaus et al., EMBO J. 8, 1989, 2445), the phosphorybosylphyrophoshate amido transferase promoter of Glycine max (gene bankaccession No. U87999) or the noden specific promoter described in EP-A-0249 676. Particularly advantageous promoters are promoters which allowthe expression in tissues which are involved in the fatty acidbiosynthesis. Most particularly advantageous are seed specific promoterssuch as usp-, LEB4-, phaseolin or napin promoter. Additionalparticularly advantageous promoters are seed specific promoters whichcan be used for monokotyledones or dikotyledones are described in U.S.Pat. No. 5,608,152 (napin promoter from rapeseed), WO 98/45461(phaseolin promoter from Arobidopsis), U.S. Pat. No. 5,504,200(phaseolin promoter from Phaseolus vulgaris), WO 91/13980 (Bce4 promoterfrom Brassica), Baeumlein et al., Plant J., 2, 2, 1992: 233-239 (LEB4promoter from leguminosa) said promoters are useful in dikotyledones.The following promoters are useful for example in monokotyledones lpt-2-or lpt-1-promoter from barley (WO 95/15389 and WO 95/23230), hordeinpromoter from barley and other useful promoters described in WO99/16890.

[0027] It is possible in principle to use all natural promoters withtheir regulatory sequences like those mentioned above for the novelprocess. It is also possible and advantageous in addition to usesynthetic promoters.

[0028] The gene construct may, as described above, also comprise furthergenes which are to be inserted into the organisms. It is possible andadvantageous to insert and express in host organisms regulatory genessuch as genes for inducers, repressors or enzymes which intervene bytheir enzymatic activity in the regulation, or one or more or all genesof a biosynthetic pathway. These genes can be heterologous or homologousin origin. The inserted genes may have their own promoter or else beunder the control of the promoter of the sequence SEQ ID No. 1 or itshomologs.

[0029] The gene construct advantageously comprises, for expression ofthe other genes present, additionally 3′ and/or 5′ terminal regulatorysequences to enhance expression, which are selected for optimalexpression depending on the selected host organism and gene or genes.

[0030] These regulatory sequences are intended to make specificexpression of the genes and protein expression possible as mentionedabove. This may mean, depending on the host organism, for example thatthe gene is expressed or overexpressed only after induction, or that itis immediately expressed and/or overexpressed.

[0031] The regulatory sequences or factors may moreover preferably havea beneficial effect on expression of the introduced genes, and thusincrease it. It is possible in this way for the regulatory elements tobe enhanced advantageously at the transcription level by using strongtranscription signals such as promoters and/or enhancers. However, inaddition, it is also possible to enhance translation by, for example,improving the stability of the mRNA.

[0032] In addition the inventive gene construct preferably comprisesadditional gene of different biochemical pathways for example genes forthe synthesis of vitamins, carotinoids, sugars such as monosaccharides,oligosaecharides or polysaccharides or fatty acid biosynthesis genes,more preferably the gene construct comprises fatty acid biosynthesisgenes such as desaturases, hydroxylases, Acyl-ACP-thioesterases,elongases, acetylenases, synthesases or reductases such as n19-, n17-,n15-, n12-, n9-, n8-, n6-, n5-, n4-desaturases, hydroxylases, elongases,n12-acetylenase. Acyl-ACP-thioesterasen, β-Ketoacyl-ACP-synthases orβ-Ketoacyl-ACP-reductases.

[0033] According to a yet further aspect of the invention there isprovided a vector comprising the nucleic acid of the present inventionor a gene construct of the present invention.

[0034] This aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a fatty acidtransporter (or a portion thereof). As used herein, the term “vector”refers to a nucleic acid molecule capable of transporting anothernucleic acid to which it has been linked. One type of vector is a“plasmid”, which refers to a circular double stranded DNA loop intowhich additional DNA segments can be ligated. Another type of vector isa viral vector, wherein additional DNA segments can be ligated into theviral genome. Certain vectors are capable of autonomous replication in ahost cell into which they are introduced (e.g., bacterial vectors havinga bacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” can be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions.

[0035] The recombinant expression vectors of the invention comprise atleast one inventive nucleic acid or at least one inventive geneconstruct of the invention in a form suitable, for expression of thenucleic acid in a host cell, which means that the recombinant expressionvectors include one or more regulatory sequences, selected on the basisof the host cells to be used for expression, which is operatively linkedto the nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory sequence(s)in a manner which allows for expression of the nucleotide sequence arefused to each other so that both sequences fulfil the proposed functionaddicted to the sequence used. (e.g., in an in vitrotranscription/translation system or in a host cell when the vector isintroduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990) or see:Gruber and Crosby, in: Methods in Plant Molecular Biology andBiotechnolgy, CRC Press, Boca Raton, Fla., eds.: Glick and Thompson,Chapter 7, 89-108 including the references therein. Regulatory sequencesinclude those which direct constitutive expression of a nucleotidesequence in many types of host cell and those which direct expression ofthe nucleotide sequence only in certain host cells or under certainconditions. It will be appreciated by those skilled in the art that thedesign of the expression vector can depend on such factors as the choiceof the host cell to be transformed, the level of expression of proteindesired, etc. The expression vectors of the invention can be introducedinto host cells to thereby produce proteins or peptides, includingfusion proteins or peptides, encoded by nucleic acids as describedherein (e.g., fatty acid transporters, mutant forms of fatty acidtransporters, fusion proteins, etc.).

[0036] The recombinant expression vectors of the invention can bedesigned for expression of fatty acid transporters in prokaryotic oreukaryotic cells, preferably in eukaryotic cells. For example, fattyacid transporter genes can be expressed in bacterial cells such as C.glutamicum, insect cells (using baculovirus expression vectors), yeastand other fungal cells (see Romanos, M. A. et al. (1992) Foreign geneexpression in yeast: a review, Yeast 8: 423488; van den Hondel, C. A. M.J. J. et al. (1991) Heterologous gene expression in filamentous fungi,in: More Gene Manipulations in Fungi, J. W. Bennet & L. L. Lasure, eds.,p. 396-428: Academic Press: San Diego; and van den Hondel, C. A. M. J.J. & Punt, P. J. (1991) Gene transfer systems and vector development forfilamentous fungi, in: Applied Molecular Genetics of Fungi, Peberdy, J.F. et al., eds., p. 1-28, Cambridge University Press: Cambridge), algae(Falciatore et al., 1999, Marine Biotechnology.1, 3:239-251), ciliatesof the types: Holotrichia, Peritrichia, Spirotrichia, Suctoria,Tetrahymena, Paramecium, Colpidium, Glaucoma, Platyophrya, Potomacus,Pseudocohnilembus, Euplotes, Engelmaniella, and Stylonychia, especiallyof the genus Stylonychia lemnae with vectors following a transformationmethod as described in WO9801572 and multicellular plant cells (seeSchmidt, R and Willmitzer, L. (1988), High efficiency Agrobacteriumtumefaciens-mediated transformation of Arabidopsis thaliana leaf andcotyledon explants, Plant Cell Rep.: 583-586); Plant Molecular Biologyand Biotechnology, C Press, Boca Raton, Florida, chapter 6/7, S.71-119(1993); F. P. White, B. Jenes et al., Techniques for Gene Transfer, in:Transgenic Plants, Vol. 1, Engineering and Utilization, eds.:Kung und R.Wu, Academic Press (1993), 128-43; Potrykus, Annu. Rev. Plant Physiol.Plant Molec. Biol. 42 (1991), 205-225 (and references cited therein) ormammalian cells. Suitable host cells are discussed further in Goeddel,Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990). Alternatively, the recombinant expressionvector can be transcribed and translated in vitro, for example using T7promoter regulatory sequences and 17 polymerase.

[0037] Expression of proteins in prokaryotes is most often carried outwith vectors containing constitutive or inducible promoters directingthe expression of either fusion or non-fusion proteins. Fusion vectorsadd a number of amino acids to a protein encoded therein, usually to theamino terminus of the recombinant protein but also to the C-terminus orfused within suitable regions in the proteins. Such fusion vectorstypically serve three purposes: 1) to increase expression of recombinantprotein; 2) to increase the solubility of the recombinant protein; and3) to aid in the purification of the recombinant protein by acting as aligand in affinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognitionsequences, include Factor Xa, thrombin and enterokinase.

[0038] Typical fusion expression vectors include pGEX (Pharmacia BiotechInc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (NewEngland Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.)which fuse glutathione S-transferase (GST), maltose E binding protein,or protein A, respectively, to the target recombinant protein. In oneembodiment, the coding sequence of the fatty acid transporter is clonedinto a pGEX expression vector to create a vector encoding a fusionprotein comprising, from the N-terminus to the C-terminus, GST-thrombincleavage site-X protein. The fusion protein can be purified by affinitychromatography using glutathione-agarose resin. Recombinant fatty acidtransporter unfused to GST can be recovered by cleavage of the fusionprotein with thrombin.

[0039] Examples of suitable inducible non-fusion E. coli expressionvectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d(Studier et al., Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1990) 60-89). Target gene expressionfrom the pTrc vector relies on host RNA polymerase tanscription from ahybrid trp-lac fusion promoter. Target gene expression from the pET 11dvector relies on transcription from a T7 gn10-lac fusion promotermediated by a coexpressed viral RNA polymerase (T7 gn1). This viralpolymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from aresident 1 prophage harboring a T7 gn1 gene under the transcriptionalcontrol of the lacUV 5 promoter.

[0040] Other vectors which are useful in prokaryotic organisms are knownby a person skilled in the art such vectors are for example in E. colipLG338, pACYC184, pBR-series such as pBR322, pUC-series such as pUC18 orpUC19, M113 mp-series, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24,pLG200, pUR90, pIN-III113-B1, lgt11 or pBdCI in Streptomyces pIJ101,pIJ364, pIJ702 or pIJ361, in Bacillus pUB110, pC194 or pBD214, inCorynebacterium pSA77 or pAJ667.

[0041] One strategy to maximize recombinant protein expression is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in the bacterium chosen for expression, such asC. glutamicum (Wada et al. (1992) Nucleic Acids Res. 20:2111-2118). Suchalteration of nucleic acid sequences of the invention can be carried outby standard DNA synthesis techniques.

[0042] In another embodiment, the fatty acid transporter expressionvector is a yeast expression vector. Examples of vectors for expressionin yeast S. cerivisae include pYepSec1 (Baldari, et al., (1987) Embo J.6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88(Schultz et al., (1987) Gene 54:113-123), and pYES2 (InvitrogenCorporation, San Diego, Calif.). Vectors and methods for theconstruction of vectors appropriate for use in other fungi, such as thefilamentous fungi, include those detailed in: van den Hondel, C. A. M.J. J. & Punt, P. J. (1991) “Gene transfer systems and vector developmentfor filamentous fungi, in: Applied Molecular Genetics of Fungi, J. F.Peberdy, et al., eds., p. 1-28, Cambridge University Press: Cambridge orin: More Gene Manipulations in Fungi [J. W. Bennet & L. L. Lasure, eds.,p. 396-428: Academic Press: San Diego]. Additional useful yeast vectorsare for example 2 mM, pAG-1, YEp6, YEp13 or pEMBLYe23.

[0043] Alternatively, the fatty acid transporter of the invention can beexpressed in insect cells using baculovirus expression vectors.Baculovirus vectors available for expression of proteins in culturedinsect cells (e.g., Sf 9 cells) include the pAc series (Smith et al.(1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow andSummers (1989) Virology 170:31-39).

[0044] The above mentioned vectors are only a small overview of possibleuseful vectors.

[0045] Additional plasmids are well known by the skilled artisan and aredescribed for example in: Cloning Vectors (Eds. Pouwels P. H. et al.Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018).

[0046] In another embodiment, the fatty acid transporter of theinvention may be expressed in unicellular plant cells (such as algae)see Falciatore et al., 1999, Marine Biotechnology.1 (3):239-251 andreferences therein and plant cells from higher plants (e.g., thespermatophytes, such as crop plants). Examples of plant expressionvectors include those detailed in: Becker, D., Kemper, E., Schell, J.and Masterson, R. (1992) “New plant binary vectors with selectablemarkers located proximal to the left border”, Plant Mol. Biol. 20:1195-1197; and Bevan, M. W. (1984) “Binary Agrobacterium vectors forplant transformation, Nucl. Acid. Res. 12: 8711-8721; Vectors for GeneTransfer in Higher Plants; in: Transgenic Plants, Vol. 1, Engineeringand Utilization, eds.: Kung und R Wu, Academic Press, 1993, S. 15-38.

[0047] A plant expression cassette preferably contains regulatorysequences capable to drive gene expression in plants cells and which areoperably linked so that each sequence can fulfil its function such astermination of transcription such as polyadenylation signals. Preferredpolyadenylation signals are those originating from Agrobacteriumtumefaciens t-DNA such as the gene 3 known as octopine synthase of theTi-plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984), 835 ff) orfunctional equivalents thereof but also all other terminatorsfunctionally active in plants are suitable.

[0048] As plant gene expression is very often not limited ontranscriptional levels a plant expression cassette preferably containsother operably linked sequences like translational enhancers such as theoverdrive-sequence containing the 5′-untranslated leader sequence fromtobacco mosaic virus enhancing the protein per RNA ratio (Gallie et al1987, Nucl. Acids Research 15:8693-8711).

[0049] Plant gene expression has to be operably linked to an appropriatepromoter conferring gene expression in a timely, cell or tissue specificmanner. Preferrred are promoters driving constitutitive expression(Benfey et al., EMBO J.8 (1989) 2195-2202) like those derived from plantviruses like the 35S CAMV (Franck et al., Cell 21(1980) 285-294), the19s CaMV (see also U.S. Pat. No. 5,352,605 and WO 84/02913) or plantpromoters like those from Rubisco small subunit described in U.S. Pat.No. 4,962,028.

[0050] Additionally vATPase-gene promoters such as an 1153 basepairfragment from Beta-vulgaris (Plant Mol Biol 1999, 39:463-475) can beused to drive ASE gene expression alone or in combination with otherPUFA biosynthesis genes.

[0051] Other preferred sequences for use operable linkage in plant geneexpression cassettes are targeting-sequences necessary to direct thegene-product in its appropriate cell compartment (for review seeKermode, Crit. Rev. Plant Sci. 15, 4 (1996), 285-423 and referencescited therin) such as the vacuole, the nucleus, all types of plastidslike amyloplasts, chloroplasts, chromoplasts, the extracellular space,mitochondria, the endoplasmic reticulum, oil bodies, peroxisomes andother compartments of plant cells.

[0052] Plant gene expression can also be facilitated via a chemicallyinducible promoter (for rewiew see Gatz 1997, Annu. Rev. Plant Physiol.Plant Mol. Biol., 48:89-108).

[0053] Chemically inducible promoters are especially suitable if geneexpression is wanted to occur in a time specific manner. Examples forsuch promoters are a salicylic acid inducible promoter (WO 95/19443), atetracycline inducible promoter (Gatz et al., (1992) Plant J. 2,397-404) and an ethanol inducible promoter (WO 93/21334).

[0054] Also promoters responding to biotic or abiotic stress conditionsare suitable promoters such as the pathogen inducible PRP1-gene promoter(Ward et al., Plant. Mol. Biol. 22 (1993), 361-366), the heat induciblehsp80-promoter from tomato (U.S. Pat. No. 5,187,267), cold induciblealpha-amylase promoter from potato (WO 96/12814) or the wound-induciblepinII-promoter (EP-A-0 375 091).

[0055] Especially those promoters are preferred which confer geneexpression in tissues and organs where lipid and oil biosynthesis occursin seed cells such as cells of the endosperm and the developing embryo.Suitable promoters are the napin-gene promoter from rapeseed (U.S. Pat.No. 5,608,152), the USP-promoter from Vicia faba (Baeumlein et al., MolGen Genet, 1991, 225 (3):459-67), the oleosin-promoter from Arabidopsis(WO 98/45461), the phaseolin-promoter from Phaseolus vulgaris (U.S. Pat.No. 5,504,200), the Bce4-promoter from Brassica (WO 91/13980) or thelegumin B4 promoter (LeB4; Baeumlein et al., 1992, Plant Journal, 2(2):233-9) as well as promoters conferring seed specific expression inmonocot plants like maize, barley, wheat, rye, rice etc. Suitablepromoters to note are the lpt2 or lpt1-gene promoter from barley (WO95/15389 and WO 95/23230) or those described in WO 99/16890 (promotersfrom the barley hordein-gene, the rice glutelin gene, the rice oryzingene, the rice prolamin gene, the wheat gliadin gene, wheat glutelingene, the maize zein gene, the oat glutelin gene, the Sorghumkasirin-gene, the rye secalin gene).

[0056] Also especially suited are promoters that confer plastid-specificgene expression as plastids are the compartment where precursors andsome end products of lipid biosynthesis are synthesized. Suitablepromoters such as the viral RNA-polymerase promoter are described in WO95/16783 and WO 97/06250 and the cIpP-promoter from Arabidopsisdescribed in WO 99/46394.

[0057] Another aspect of the invention pertains to host cells into whicha recombinant expression vector of the invention has been introduced.The terms “host cell” and “recombinant host cell” are usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

[0058] Vector DNA can be introduced into prokaryotic or eukaryotic cellsvia conventional transformation or transfection techniques. As usedherein, the terms “transformation” and “transfection”, conjugation andtransduction are intended to refer to a variety of art-recognizedtechniques for introducing foreign nucleic acid (e.g., DNA) into a hostcell, including calcium phosphate or calcium chloride co-precipitation,DEAE-dextran-mediated transfection, lipofection, natural competence,chemical-mediated transfer, or electroporation. Suitable methods fortransforming or transfecting host cells including plant cells can befound in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd,ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1989) and other laboratory manuals such asMethods in Molecular Biology, 1995, Vol. 44, Agrobacterium protocols,ed: Gartland and Davey, Humana Press, Totowa, N.J.

[0059] The vector is introduced into a microorganism or plant cell(e.g., via Agrobacterium mediated gene transfer, biolistictransformation, polyethyleneglycol or other applicable methods) andcells in which the introduced ASE gene has homologously recombined withthe endogenous fatty acid transporter gene are selected, using art-knowntechniques. In case of plant cells the AHAS gene described in Ott etal., J. Mol. Biol. 1996, 263:359-360 is especially suitable for markergene expression and resistance towards imidazolinone or sulphonylureatype herbicides.

[0060] In another embodiment, recombinant organisms such asmicroorganisms can be produced which contain selected systems whichallow for regulated expression of the introduced gene. For example,inclusion of a fatty acid transporter gene on a vector placing it undercontrol of the lac operon permits expression of the fatty acidtransporter gene only in the presence of IPTG. Such regulatory systemsare well known in the art. Recombinant organisms means an organism whichcomprises an inventive nucleic acid sequence, a gene construct or avector in the cell or inside the genome at an place which is not the“natural” place or an the “natural” place but modified in a manner whichis not the natural manner that means the coding sequence is modifiedand/or the regulatory seqeunce is modified. Modified means that a singlenucleotide or one or more codons are changed in comparison to thenatural sequence.

[0061] A host cell of the invention, such as a prokaryotic or eukaryotichost cell in culture, can be used to produce (i.e., express) a fattyacid transporter. An alternate method can be applied in addition inplants by the direct transfer of DNA into developing flowers viaelectroporation or Agrobacterium medium gene transfer. Accordingly, theinvention further provides methods for producing fatty acid transportersusing the host cells of the invention. In one embodiment, the methodcomprises culturing the host cell of invention (into which a recombinantexpression vector encoding a fatty acid transporter has been introduced,or into which genome has been introduced a gene encoding a wild-type oraltered fatty acid transporter) in a suitable medium until fatty acidtransporter is produced. In another embodiment, the method furthercomprises isolating fatty acid transporters from the medium or the hostcell.

[0062] Host organisms suitable in principle to cover the nucleic acid ofthe invention, the novel gene construct or the inventive vector are allprokaryotic or eukaryotic organisms. The host organisms advantageouslyused are organisms such as bacteria, fungi, yeasts, animal or plantcells. Additional advantageously organisms are Fungi, yeasts or plants,preferably fungi or plants, very particularly preferably plants such asoilseed plants containing high amounts of lipid compounds such asrapeseed, primrose, canola, peanut, linseed, soybean, sufflower,sunflower, borage or plants such as maize, wheat, rye, oat, triticale,rice, barley, cotton, manihot, pepper, tagetes, solanaceaous plants suchas potato, tobacco, eggplant, and tomato, Vicia species, pea, alfalfa,bushy plants (coffee, cacao, tea), Salix species, trees (oil palm,coconut) and perennial grasses and forage crops. Particularly preferredplants of the invention are oilseed plants such as soybean, peanut,rapeseed, canola, sunflower, safflower, trees (oil palm, coconut).

[0063] According to a further aspect of the invention there is provideda nucleic acid molecule comprising SEQ ID NO:1 or a part thereof, orhomologue thereof, which encodes a peroxisomal ABC (ATP-bindingCassette) transporter.

[0064] Also provided is a nucleic acid encoding an ABC cassettetransporter protein especially one involved in fatty acid transportacross peroxisomal membranes, the nucleic acid being selected from agroup consisting of:

[0065] (a) DNA having the nucleotide sequence given herein in SEQ ID NO:1 which encodes a protein having the amino acid sequence given herein asSEQ ID NO:2.

[0066] (b) nucleic acid which hybridize to DNA of (a) above (e.g. understringent conditions); and

[0067] (c) nucleic acids which differ from the DNA of (a) or (b) abovedue to the degeneracy of the genetic code, and which encodes a proteinencoded by a DNA of (a) or (b) above.

[0068] DNAs of the present invention include those coding for proteinshomologous to, and having essentially the same biological properties as,the proteins disclosed herein, and particularly the DNA disclosed hereinas SEQ ED NO: land encoding the protein given herein SEQ ID NO:2. Thisdefinition is intended to encompass natural allelic variations therein.Thus, isolated DNA or cloned genes of the present invention can be ofany plant species of origin. Thus, DNAs which hybridise to DNA disclosedherein as SEQ ID NO:1 (or fragments or derivatives thereof which serveas hybridisation probes as discussed below) and which code on expressionfor a protein associated with fatty acid or acyl CoA transport intoperoxisomes (e.g., a protein according to SEQ ID NO:2) are included inthe present invention.

[0069] According to a further aspect of the present invention there isprovided use of the nucleic acid of the invention and/or protein orpolypeptide encoded thereby in any one or more of the followingprocesses: regulating fatty acid transport across peroxisome and/orglyoxisome membranes; regulating growth; regulating seed developmentand; modulating fatty acid utilisation by the plant.

[0070] According to a yet further aspect of the present invention thereis provided a method of regulating any one or more of the followingprocesses: regulating fatty acid transport across peroxisome and/orglyoxisome membranes; regulating growth; regulating seed developmentand; modulating fatty acid utilisation by the plant comprisinggenetically engineering a plant cell or tissue or seed so as to enhanceor reduce/prevent expression of the nucleic acid of the presentinvention.

[0071] Such techniques are well-known in the art and include but are notrestricted to: reduction of expression of the nucleic acid and encodedprotein by antisense expression of part or all of the sequencecorresponding to SEQ ID No1, expression of part or all of SEQ ID No1 asdouble stranded interference RNA (RNAi); or co-supression of theendogenous gene brought about by introduction of additional copies ofpart or all of SEQ ID No1. Conversely expression may be increased, oraltered spatially and temporally, by the introduction of constructsfused to different regulatory sequences as hereinbefore described.

[0072] According to a yet further aspect of the invention there isprovided a method of modification of a plant cell to increase ordecrease the transport of some or all fatty acids across cell membranesand/or to increase or prevent their breakdown.

[0073] Preferably the modification could take the form of mutating,disabling or deleting the CTS gene. The CTS gene could also be modifiedto alter its expression levels in specific tissues or at specific times.By example and not by way of limitation, CTS expression could beinhibited in developing seeds but not in germinating seeds.Alternatively, the plant cell may be modified so as to containing anincreased number of copies of the nucleic acid according to theinvention as compared to the wild-type.

[0074] Mutation of the sequence may be through designed changes orrandom changes followed by selection to introduce variants of SEQ ID No1 and SEQ ID No2 with altered substrate specificity or transport rate orregulation, or through the expression of mutants with dominant negativeactivity.

[0075] The invention therefore includes transgenic plants comprising anucleic acid molecule of the invention as well as transgenic plantsadapted to increase or decrease expression of an active peroxisomal ABCtransporter, for example by having increased or decreased numbers ofgene copies or modification of transcription control elements

[0076] According to a yet further aspect of the invention there isprovided a method of regulating fatty acid levels in plants comprisinggenetically engineering a plant cell or tissue or seed so as to disrupt,deactivate, disable, mutate, delete, knockout or rendertranscriptionally ineffective a nucleic acid according to the invention.

[0077] According to a yet further aspect of the invention there isprovided a plant cell and/or a plant tissue and/or plant and/or plantseed that does not containing a transcriptionally activated/activatableform of the nucleic acid molecule according to the invention or containsa reduced number of copies of the nucleic acid of the present inventionas compared to the wild-type.

[0078] According to a yet further aspect of the invention there isprovided a plant generated from a plant cell and/or plant tissue and/orplant and/or plant seed which contains a disrupted, deactivated,disabled, mutated, deleted, knocked-out or rendered transcriptionallyineffective nucleic acid according to the invention as compared to thewild-type.

[0079] According to a yet further aspect of the invention there isprovided a plant cell and/or a plant tissue and/or plant and/or plantseed comprising:

[0080] (i) an increased number of copies of the nucleic acid accordingto the invention as compared to the wild-type;

[0081] (ii) increased transcription of the nucleic acid according to theinvention as compared to the wild-type; or

[0082] (iii) a differing number of copies of the nucleic acid accordingto the invention depending on the time of development of the plant.

[0083] According to a yet further aspect of the invention there isprovided a plant generated from a plant cell and/or plant tissue and/orplant and/or plant seed comprising:

[0084] (i) an increased number of copies of the nucleic acid accordingto the invention as compared to the wild-type;

[0085] (ii) increased transcription of the nucleic acid according to theinvention as compared to the wild-type; or

[0086] (iii) a differing number of copies of the nucleic acid accordingto the invention depending on the time of development of the plant.

[0087] According to a yet further aspect of the invention there isprovided a primer comprising any one of SEQ ID NOS: 3 to 10 or partsthereof capable of recognising the nucleic acid of the present inventionor homologues thereof, the primer being specific for a nucleic acidaccording to the invention.

[0088] The following T-DNA knockout lines containing insertions in theCTS gene were screened from Wisconsin knockout population alpha:—

[0089] cts-2; position of insertion in genomic DNA is 16,674 inAccession AL161596 (Arabidopsis thaliana DNA chromosome 4, contigfragment No. 92, VERSION AL161596.2). This corresponds to Exon 3 of thegene or position 846 in Sequence ID NO:1 (cDNA) and in codon Thr115 inSEQ ID NO:2 (the derived amino acid sequence). The primers used in theinitial PCR screen are preferably H1A6T7 DSR1 (SEQ ID NO:8) AND JL 202(SEQ ID NO:11).

[0090] F27; position of insertion in genomic DNA is 15,873 in AccessionAL161596. This is in Intron-1 which is located in the 5′-UTR. Theprimers used in the initial PCR screen are preferably H1A6T7 DSF1 (SEQID NO:4) and JL 202(SEQ ID NO:11).

[0091] F12; position of insertion in genomic DNA is 17,318 in AccessionAL161596. This is in Intron-4. The primers used in the initial PCRscreen are preferably H1A6T7 DSF1 (SEQ ID NO:4) and JL 202(SEQ IDNO:11).

[0092] Preferably, the primers of the present invention include:

[0093] Primer H1A6T7 DSF1 Forward primer 5′ at position 15,667 ofAccession AL161596, (SEQ ID NO:4); Primer H1A6T7-DSF2 5′ position at15,640 of Accession ALI 61596, (SEQ ID NO:5); Primer H1A6T7 DSF3 5′position at 15,320 of Accession AL161596, (SEQ ID NO:6); Primer H1A6T7DSF 5′ position at 15,542 of Accession AL161596, (SEQ ID NO:3); PrimerH1A6T DSR1 Reverse primer 5′ at position 20,611 of Accession AL161596,(SEQ ID NO:8); Primer H1A6T7 DSR2 5′ position at 17,819 of AccessionAL161596, (SEQ ID NO:9); Primer H1A6T7 DSR3 5′ position at 21,191 ofAccession AL161596, (SEQ ID NO:10); Primer H1A6T7 DSR 5′ position at19,978 of Accession AL161596 (SEQ ID NO:7) and/or, Primer JL 202 LeftBorder primer for PCR out of constructs of Wisconsin knockout populationalpha (SEQ ID NO:11).

[0094] According to a yet further aspect of the invention there isprovided use of a primer comprising any one of SEQ ID-NOS: 3 to 10 orparts thereof capable of recognising the nucleic acid of the presentinvention or homologues thereof, in identifying a nucleic acid sequenceaccording to the invention.

[0095] According to a yet further aspect of the invention there isprovided a method of identifying plant material selected from a plantcell and/or plant tissue and/or plant and/or plant seed comprising adisrupted, deactivated, disabled, mutated, deleted, knocked-out orrendered transcriptionally ineffective nucleic acid according to theinvention comprising contacting the plant material with a primercomprising any one of SEQ ID NOS: 3 to 10.

BRIEF DESCRIPTION OF THE FIGURES

[0096]FIG. 1 illustrates alignment of peroxisomal ABC transporters. Thepeptide sequences of Human ALD and PMP70 were aligned with ArabidopsisCTS and yeast Pxa1, CTS. Although the arabidopsis ABC is a doubletransporter consisting of 2 sets of trasmembrane domains comprisingmultiple transmembrane helicesand 2 ATPase domains (format: TMdomain-ATPase-TM domain-ATPase) its closest homologs outside the plantkingdom are half transporters (format: TM domain-ATPase). For thisalignment the peptide sequence of the Arabidopsis abc was arbitrarilydivided into two halves: CTSa and CTSb. Conserved domains previouslydescribed in the literature are highlighted. Loop 1 is conserved amongperoxisomal ABC transporters only. The EAA-like motif, Walker A and B,and the C-sequence are all conserved between prokaryotic and eukaryoticABC transporters.

[0097]FIG. 2 illustrates analysis of CTS transcript levels duringgermination in the light. Seeds were surface sterilized using Milton®and imbibed in the dark at 4C for 4d before plating and incubating at22C. Data points are days post imbibition (dpi).

[0098]FIG. 3 shows expression and purification of second ATPase domainof CTS.

[0099]FIG. 4 illustrates the location of CTS to membrane fractions.Arabidopsis cell suspension culture derived from leaf was homogenizedand centrifuged at low speed (1000 g) to remove nuclei followed by 20000 g to sediment membrane fractions. These were then resuspended andloaded onto a sucrose step gradient Fractions of equivalent proteinloading were separated by SDS-PAGE followed by immuno-blotting withantisera raised against CTS. Lane 1, SDS-extraction of originalhomogenate; lane 2, post-nuclear supernatant; lane 3, resuspendedmembrane pellet; lane 4, membrane recovered at 0.5 M/1.6M sucroseinterface and lane 5, 1.6M/2.2M sucrose interface. Concentration of CTSin the membrane fractions is consistent with CTS being a membraneprotein.

[0100]FIG. 5 represents the location of T-DNA insertions in three CTSknockouts cts-2F27 and F12. Exons are shown as dark shaded boxes. Noncoding transcribed regions are indicated by light shading.

[0101]FIG. 6 illustrates the isolation of individual CTS knockoutplants. 100 individual plants were screened by PCR for the exon 2knockout (cts-2) from a pool containing seed from 9 T-DNA tagged linespreviously identified by PCR. PCR was carried out using a T-DNA specificprimer (JL-202) and gene-specific primer DSR1 (A) or DSR3 (B). Apositive plant (lane 5) was identified from this sample of 15individuals. (C) HindM restriction digest of genomic DNA from wt(lane 1) and the positive plant (lane 2) was probed with CTS probe.Bands of 2.5 kb and 6.5 kb correspond to predicted sizes for wt CTS. Theband of 8.7 kb corresponds to a predicted size consistent with a T-DNAinsertion in exon 2. Together these data suggest that the positive plantis a heterozygote having one copy of the wt CTS gene and a mutant genecontaining a T-DNA insertion. (I) PCR identifying presence of knockoutplants in a pool of germinating seedlings from 9 T-DNA tagged lines forknockout F27 (lanes 1-3) and knockout F12 (lanes 66) using a T-DNAspecific primer and a range of gene-specific primers.

[0102]FIG. 7 illustrates CTS localisation in peroxisomes. Sucrosedensity gradient showing the localisation of peroxisomal markerscatalase (closed circles) and 3-ketoacyl thioloase (KAT), ER markercalreticulin (CAL), mitochondrial marker adenine nucleotide translocator(ANT) and chlrophyll (open circles). CTS antigen follows thedistribution of catalase and thiolase.

[0103]FIG. 8 illustrates the profiles of triacyl glycerol-derived fattyacids and acyl CoA levels in wild type and cts-2 mutant: a=Comparison ofTAG-derived fatty acids in the wild types and cts-2 mutant in seeds andseedlings 0, 2 and 5 days after sowing; b=profile of TAG-derived fattyacids in imbibed wild type and mutant seeds by chain length; c=as b butprofile from 5 day old seedlings; d=comparison of Acyl CoA levels inwild types and cts-2 mutant seedlings 0,2 and 5 days after sowing;e=profile of Acyl CoAs in imbibed wild type and mutant seeds by chainlength and: f as e but profile from 5 day old seedlings.

DETAILED DESCRIPTION OF THE INVENTION

[0104] The yeast and human peroxisomal ABC transporter proteins wereused to search the publically accessible sequence databases ofArabidopsis taliana using the BLAST Algorithm One sequence wasidentified (T5J17.20; AT4G39850, from chromosome IV) which had asignificant sequence homology to all the probe sequences and whichcorresponded to an EST (H1A677) from a 3 day seedling hypocotyl library.This clone was obtained from the Arabidopsis Biological Resource Centreat Ohio State University (USA), fully sequenced and named CTS (SEQ IDNO:1).

[0105] Bioinformatic analysis of the sequence revealed that this encodeda protein of 1337 amino acids with a predicted molecular weight of149,576 (SEQ ID NO:2). Functional ABC transporters have 4 domains, twosets of 5 or 6 transmembrane spans and two ATPase domains which can beon separate polypeptide chains or fused in various combinations. CTS isof the type in which all domains are fused whereas ScPxa2p is of thehalf transporter type containing one transmembrane domain and one ATPasedomain. CTS contains all the conserved residues typical of an ABCtransporter. The two half transporter domains of CTS are 34% identicalto one another. The highest matches on BLASTp search (P<7.1e-67) are themammalian PMP70 and adrenoleukodystrophy proteins, also halftransporters that are involved in fatty acid transport into peroxisomes.Saccharomyces cerevisiae Pxa2p is the 6^(th) most similar sequence(P=1.5e-44). An alignment of the deduced amino acid sequences of CTSwith PMP70, ALDP, ScPxa1p and ScPxa2p is shown in FIG. 1. The ATPbinding sites, designated Walker A and B motifs are highly conserved inall ATPases. The C-sequence, otherwise known as the ABC motif isdiagnostic of the ABC transporter superfamily, as is the EAA sequence.The sequence NSEEIAFY is diagnostic of the human and yeast peroxisomalABC transporters and the closely related sequence H(S/A) SIAF(Y/F)occurs in the two halves of CTS. The loop1 region and the sequencePQRPY(M/T)(A/C)LGTLRDQ is diagnostic of animal peroxisomal ABCtransporters. The almost identical sequence PQRPY(M/T)(A/C)LGTLRDQoccurs in both halves of CTS. Therefore CTS contains sequences whichassign it to the peroxisomal sub-class of ABC transporters.

[0106] To determine whether the expression, pattern of CTS wasconsistent with its involvement in fatty acid uptake in peroxisomes, anorthern blot was performed (FIG. 2). Fatty acid oxidation is anon-going process in plant cells due to turn over of membrane lipids byβ-oxidation. However higher levels of expression would be expectedduring and immediately after germination as is seen for the β-oxidationenzymes thiolase (Germain et al., 2001) and acyl CoA oxidase (Hooks etal., 1999). Consistent with this the CTS probe detects a transcript of4.9 kb that is expressed throughout the first 12 days post imbibitionbut with highest expression at day1.

[0107] To provide experimental evidence for the peroxisomal location ofCTS, antibodies were raised to the second ATPase domain. A fragmentcorresponding to amino acids 1112-1337 was cloned into pET28b (Novagen)to produce a his tagged recombinant protein. The recombinant protein,which is in inclusion bodies was purified by NTA agarose chromatography(FIG. 3). Affinity purified antisera detected a protein of ca.140 kDa inmembrane fractions from Arabidopsis seedlings and tissue culture cells(FIG. 4). Further experiments demonstrated the localisation of thisprotein specifically in peroxisomes/glyoxysomes (FIG. 7).

[0108] Thus far the function of the CTS protein is inferred fromsequence homology to known peroxisomal fatty acid ABC transporters. Toobtain functional information for CTS knockout mutations in Arabidopsiswere sought through reverse genetics. Primers were designed and sent tothe Arabidopsis knock out facility at the University of Wisconsin (USA)to screen their population of T-DNA tagged mutants. Three alleles of CTSwere detected (FIG. 5). The location of the T-DNA insertions weredetermined by sequencing the PCR products. T-DNA insertions are in exon2 and introns 1 (in the 5′UTR) and 4 of CTS. The insertions in exon 2 ofCTS would be predicted to be a null alleles, while those in the intronsmay or may not give a phenotype depending on whether they are correctlyspliced out from the transcript. Single heterozygous plants have beenobtained for each T-DNA insertion (FIG. 6). Heterozygous cts-2 plantswere allowed to self fertilise and homozygous progeny selected.Homozygous seeds and seedlings were subjected to triacyl glycerol andacyl CoA profiling (FIG. 8). These results provide strong support forthe proposed biochemical function of CTS as a fatty acyl CoAtransporter.

[0109] Methods

[0110] The cts-2 mutant allele was obtained by PCR-based screening ofthe Wisconsin-alpha gene knockout lines for insertions in the CTS gene(Krysan et al., 1999). The sequence of the CTS RNA was obtained usingthe cDNA clone H1A6T7 (ABRC, Ohio State Univ.). Total RNA was extracted,separated by denaturing agarose gel electrophoresis and transferred to ahybridisation membrane. The membrane was hybridised with 32P-labelledclone H1A6T7 as described by Hooks et al., (1999) and detected byphophoimaging.

[0111] A polyclonal antibody was raised against a C-terminal fragment ofCTS (amino acids 1112-1337) expressed in E. coli strain BL21(DE3)pLysS(Novagen) using a pET28b (Novagen) construct containing an NheI-BamHIfragment of cDNA clone H1A6T7 (EMBL accession AJ311341). The antibodywas affinity-purified using the recombinant fragment (Tugal et al.,1999).

[0112] Acyl CoAs and total lipids were extracted from five replicate3-10 mg tissue samples and analyzed according to Larson and Graham(2001). An aliquot of the total lipid extract was used fortriacylglycerol (TAG) determination. A 1 mL 100 mg bed volume Bond-Elut(Varian, Surrey, UK) SPE column was prepared by elution with 2×1 mLmethanol, 3×1 mL hexane, and then 100 μL sample loaded in hexane. TAGswere eluted with 1.5 mL 2:3 (v/v) chloroform:hexane, dried under vacuum,transmethylated to fatty acid methyl esters, and analyzed as describedpreviously (Larson and Graham, 2001). Specificity for TAG separation wasoptimized so that the diacylglycerol, dipalmitin (Sigma) was excludedfrom the SPE eluate.

[0113] To provide further information on the specific nature of theblock in lipid breakdown and hence the function of the CTS protein, thelevels of triacyl glycerol (TAG) and acyl CoAs were measured in thects-2 mutant and corresponding WS wild type. All lines were germinatedin the presence of 1% sucrose (and the testa of the cts-2 mutants wasruptured to allow germination) to ensure that seedlings were at similarmorphological stages of development The summed changes in fatty acidcontent of extracted TAG indicate similar levels of TAG-derived fattyacids in imbibed seeds of the wild type and mutant on day 0 (FIG. 8a).Higher apparent TAG levels per seed(ling) in the mutant reflects biggerseed size and is not seen when the data is expressed on a fresh weightbasis). TAG fatty acid levels only declined slightly after 2 daysgermination even in wild type seedlings, presumably due to the presenceof sucrose as an alternative energy source. However by day 5, TAGderived fatty acids had decreased by 95.8% in the wild types but by only32.3% in the mutant All TAG-derived fatty acid chain lengths weremobilised after 5 days germination for wild type, but the cts-2 mutantretained high levels of the same TAGs (FIG. 8b,c).

[0114] Total Acyl CoAs increased in both lines over the period 05 days(FIG. 8d), but this is much more dramatic in the mutant By day 5 some ofthe increase in both wild type and mutant seedlings may reflect newlipid synthesis (e.g. for membrane biogenesis). However the moststriking observation is the retention of 20:1 and to a lesser extent20:0 and 22:1 CoA's in seedlings of the cts-2 mutant (FIG. 8e,f). As C20fatty acids are only very minor components of non-storage lipids inArabidopsis, these data demonstrate that there is a severe block incarbon flux from stored triacyl glycerols during germination in themutant.

[0115] Analysis of TAG-derived fatty acids and acyl CoAs demonstratesthat while some lipid is mobilised in the cts-2 mutant, catabolism isinhibited before β-oxidation, resulting in an increased acyl CoA pool.This is particularly pronounced for C20 and C₂₋₂ acyl CoAs which arepredominantly T-G-derived. These data argue strongly that the primarydefect in the cts-2 mutant is in transport of fatty acyl CoAs intoperoxisomes. 18:2 and 18:3 CoAs do not accumulate to a similar extent,which may reflect their use in the synthesis of structural lipids by ERmediated pathways. In contrast C20:1 is not a component of structurallipids and may accumulate because it lacks a synthetic sink route. It ispossible that the accumulation of 20:1 CoA, or depletion of freecoenzyme A results in the inhibition of lipolysis and therefore therelease of further fatty acids from storage TAG. The accumulation ofacyl CoAs argues that these are the substrates of the CTS protein, andsuggests that unlike X-ALD patients CTS mutants retain VLCFA synthetaseactivity. The substantial accumulation of long and very long chain acylCoA's in the cts-2 mutant is consistent with the activation of thesefatty acids by acyl CoA synthetases on the cytoplasmic side of theperoxisomal membrane, as reported for S. cerevisiae (Hettema et al.,1996), mammals (Mannaerts et al., 1982) and plants (Olsen and Lusk1994). The finding that all fatty acid chain lengths are mobilized inwild type and retained in the mutant argues that the transporter hasbroad substrate specificity with respect to acyl chain length. AcylCoA's are amphipathic molecules, as are the substrates for many ABCtransporters.

[0116] Oilseeds can be engineered to produce economically valuableunusual fatty acids. However, the exact fate of the unusual fatty acid,once it is made, is not known What is known is that not enough of thevaluable fatty acid ends up in seed oil. One factor that appears tolimit novel oil yield is that the fatty acids comprising them are brokendown before being incorporated into oil (Eccleston and Ohlrogge 1998).CTS, the nucleic acid of the present invention, is an excellentcandidate to be involved at the beginning of this process. As indicatedby the data in FIG. 8, mutating the function of CTS reduces or abolishthe entry of some or all fatty acids into the glyoxisome/peroxisome andtherefore prevent their breakdown We predict this would allow theiraccumulation in seeds or other plant tissues. However, the plant stillneeds to be able to utilise endogenous fatty acids for germination andgrowth. Blocking β-oxidation through the mutation of thiolase results ingermination but subsequent growth is dependent on exogenously suppliedsucrose (Germain et al., 2001). It should be possible to alter theexpression levels of CTS in specific tissues or at specific times, forexample inhibiting its activity in developing seeds but not ingerminating seeds. Alternatively if we can determine and then alter itssubstrate specificity we may be able to allow accumulation of desirednovel oils but not prevent germination and seedling establishment usingendogenous fatty acids. An additional problem is that when high levelsof novel oils can be achieved, oilseeds cannot use these foreign oils asan efficient source of fuel for young seedlings to grow, resulting innon-viable seed. For example, expressing a form of CTS that cannottransport the novel oils in the developing seed but switching on a formwhich can use the novel oil as a substrate during germination mightovercome this problem. These proteins with altered substrate specificitymight arise as a resulted of targeted or random mutation of the genefollowed by an appropriate selection, or the use of the Arabidopsisprotein to isolate homologues which may have different substratespecificities from species which naturally accumulate high levels of thedesired novel oils. Therefore the protein of the present invention hasthe potential to increase the accumulation of novel oils in seeds toeconomically viable levels.

[0117] Mutants in β-oxidation show resistance to2,4-dichlorophenoxybutyric acid (2,4 DB, Hayashi et al., 1998 andindole-3-butyric acid (IBA, Zollman et al 2000) and β-oxidation isclearly impaired in the cts-2 mutant, most likely as a consequence of adefect in transport of fatty acyl CoAs into the peroxisome formetabolism CTS maps to the same location on chromosome IV as the ped3mutant (Hayashi et al., 1998). IBA and 2,4 DB are amphipathic moleculesand CTS is a good candidate to mediate their uptake into peroxisomes. Ifa dominant negative or RNAi version of CTS could be expressed under thecontrol of a regulated promoter, it could be used as a selectable markerfor plant transformation. The expression of the dominant negative formof the protein would most likely confer resistance to IBA or 2,4 DB.Resistance results in a ‘long root’ phenotype and would allow plantsexpressing this marker to be selected on medium containing IBA or 2,4DB, sucrose and the inducing molecule for the regulated promoter. Theadvantage is that the selectable marker is of plant origin and unlike,for example, herbicide resistance, confers selection only in thepresence of three separate molecules which is not going to occur innature. It does not confer antibiotic resistance, so there can be nodanger of disseminating resistance in the environment. The use of aregulated promoter results in expression of the selection only underdefined conditions.

REFERENCES

[0118] Footitt, S., Slocombe, S P., Laarner, V., Kurup, S., Wu, Y.,Larson, T., Graham, I., Baker, A and Holdsworth (2002) EMBO 21, 12,2912-2922.

[0119] Germain, V., Rylott, E. Larson, T. R, Sherson, S. M., Bechtold,N. Carde, J-P., Bryce J. H.

[0120] Graham, I. A. and Smith, S. M. (2001) Plant J. 28, 1-12.

[0121] Hayashi M, Toriyama, K, Kondo, M. ad Nishimura, M. (1998) PlantCell 10, 183-195.

[0122] Hettema, E. H., van Roermund, C W T, Distel, B. van den Berg, M.Vilela, C., Rodrigues-Pousada C, Wanders, R J A and Tabak, H F (1996)EMBO J. 15, 3813-3822.

[0123] Hooks, M. A., Kellas, F. and Graham, I. A. (1999) Plant J. 20,1-3.

[0124] Krysan, P. J., Young, J. C. and Sussman, M. R. (1999) Plant Cell,11, 2283-2290.

[0125] Larson, T. R, Graham, I. A. (2001) Plant J. 25:115-125

[0126] Mannaerts, G. P., van Veldhoven, P., van Broekhoven, A.,Vandebroek, G., and Debeer, L. J. (1982). Biochem. J. 204, 17-23.

[0127] Mosser, J. Douar, A. M., Sarde, C-O., Kioschis, P. Feil, R Moser,H., Poustka, A-M., Mandel, J-M. and Augbourg, P (1993) Nature 361,726-730.

[0128] Olsen, J. A. and Lusk, K. R (1994) Phytochemistry 36, 7-9.

[0129] Russell, L., Larner, V., Kurup, S., Bougourd and Holdsworth, M(2000) Development, 127, 3759-3767.

[0130] Tugal, H. B., Pool, M. and Baker, A. (1999) Plant Physiology,120, 309-320.

[0131] Zollman B. K., Yoder, A. and Bartel, B. (2000) Genetics 156,1323-1337

1 11 1 5073 DNA Arabidopsis thaliana 1 atgtgctgca aggcgattaa gttgggtaacgccagggttt tcccagtcac gacgttgtaa 60 aacgacggcc agtgaattgt aatacgactcactatagggc gaattgggta ccgggccccc 120 cctcgaggtc gacggtatcg ataagcttgatatcgaattc gcggccgcct ctctctctct 180 ctatctctat ctctcgattt gggggagttccgtcacggtg gactagtacg tctcgttgcc 240 gttggtggcg tagtcggaat taatttcctcggcgttgaga ttcacatggt ctagaattct 300 agctaagtgg ttgttgttgt tgttacgatttccgatttct cgagtttttt tttttatatt 360 tagcttctgt ttcgtttatc cctcccggagacactccttg gtcgaatctc tcatgctgag 420 gtgttttgga cacttgttgt caagaagaaaccagttttgg ttctgattaa tcgttggttg 480 gaaaatatac tcaattccag gccatgccttcacttcaact attgcagtta actgagcggg 540 gtcggggtct tgtagcgtca agacggaaatctatactgct tgcggctggg attgtagctg 600 ctggtggaac tgctgtttac ctgaaatcaagggtcgcttc ccggaggcct gattcttcgc 660 gtctttgcaa tggtcagagt gatgatgatgagactttgga aaagctgact gcaactgatc 720 aaaatgcaaa gataaccacg aaaaagaagaaaggaggagg attgaagtct cttcaggttc 780 tgactgctat tcttctctct cagatgggaaaaatgggtgc cagggatctt ttggcactag 840 tcgccaccgt ggttttcaga acagctttgagcaatagatt ggcaaaagtg caaggtttcc 900 ttttccgtgc tgctttctta aggcgtgcgccactgtttct acggctcatc tccgagaata 960 ttatgttgtg tttcatgcta tcaacattgcactctacttc aaagtacata actggggcat 1020 tgagtttgcg attcagaaag atattgaccaagattatcca ttcacactat tttgagaata 1080 tggtatatta caaaatatca cacgtggatggtcggattac gcaccctgaa caaagaattg 1140 ccagcgatgt accaagattc tcctcagagttgagcgatct tatactggat gatttgacgg 1200 cggttactga tggaattttg tatgcatggcgcctgtgttc atatgctagt ccaaaataca 1260 tcttctggat actggcctat gtactgggggctgggacggc gataagaaac ttttctcctt 1320 cttttgggaa attgatgtcc aaggaacagcagttagaagg agagtaccgg caacttcatt 1380 cacgcttaag gactcattcg gaaagcatagcattctatgg tggggaaacc agggaagaat 1440 ctcatataca acaaaagttc aagaatcttgttagccatat gagtcacgtg cttcatgatc 1500 actggtggtt tggtatgatc caagattttctgctgaagta tcttggggcc acagttgcag 1560 ttattctgat tatcgaacca ttcttctctgggcatctaag acctgacgac tcgaccttag 1620 gaagagctga gatgcttagc aatataagatatcacactag tgtcattata tctctctttc 1680 aggcgttggg aacactttct ataagttccaggcggctcaa ccgactcagt ggttatgctg 1740 accgaatcca tgagttgatg gctgtctcaagagaactcag tggtgatgat aaatcgtctt 1800 tccagagaaa tagaagcaga aattatctaagtgaagctaa ttatgtagag ttttccgatg 1860 tcaaggttgt tactccaacc ggaaatgttttggtggagga tctcaccctt cgagttgagc 1920 aagggtctaa tcttctgatt acaggtcctaatggaagtgg caagagttcc cttttccgag 1980 tattaggagg tctatggccc ctggtgtctggacatattgt gaagccagga gttggttctg 2040 atcttaacaa ggagatcttc tatgtgccgcaacggcctta tatggcagta ggaacacttc 2100 gtgaccagtt aatatatcct cttacttctggccaagagag tgaactgctc actgagattg 2160 gaatggtgga gctattgaaa aatgttgatctagaatattt attggatcgc taccaacctg 2220 aaaaagaggt taattggggt gatgaattatctcttggaga gcaacagaga ttgggtatgg 2280 ccagactatt ctaccacaaa cccaaatttgcaattctaga tgaatgcaca agtgctgtca 2340 caactgatat ggaagaacgc tttgccgctaaggttcgagc tatgggaact tcttgcataa 2400 caatctccca tcgtccagcg cttgttgcattccatgatgt tgttctgtca ttagacggtg 2460 aaggaggatg gagtgttcat tacaagagggatgactctgc ccttctgacg gatgctgaaa 2520 ttgattcagt gaaaagttca gatacagatcggcaaaatga tgcgatggtt gttcaacgag 2580 cgtttgctgc agctagaaag gaatctgctactaattcaaa ggctcagtcg taccagacac 2640 agttaattgc aagatcacct gttgtagataaaagtgtagt gttgcctcgt tttcctcaac 2700 ctcaaacatc ccaaagggct ttaccatcaagagtagctgc aatgttaaac gtgttgatac 2760 ccactatatt tgacaagcaa ggagctcaactgcttgctgt tgcttgcctt gtcgtctcaa 2820 gaacgctgat ctctgaccga atagcctctttgaatgggac cactgtgaag tatgtcttgg 2880 agcaagataa ggcagccttt gttcgtttgattggtttgag tgttctccaa agtggtgcat 2940 cttctataat tgctccttca ctaaggcatttaacgcaaag gctagcgtta gggtggagga 3000 ttcgtttgac tcaacatctg ctaaggaactatttgagaaa taatgcgttt tacaaggttt 3060 tccacatgtc aggcaatagt attgatgcggaccagagact cactcgtgac ctggaaaagt 3120 taaccgctga cttgtctgga cttcttactggaatggtaaa gccatcggtt gacattctct 3180 ggttcacctg gaggatgaag ttactgactggtcagagggg agttgccata ctttacacat 3240 atatgttact tggtcttggt tttctgagacgtgttgctcc cgatttcggt gatctagccg 3300 gtgaagaaca gcagcttgaa gggaagtttcggtttatgca cgagaggctg aacactcatg 3360 ctgaatctat tgcattcttt ggaggtggagctcgagaaaa ggctatggtt gacaaaaaat 3420 tcagggccct actggatcat tctctcatgctcttgaggaa gaaatggttg tatggcatac 3480 ttgatgattt tgtgacaaag caacttcccaataatgtgac gtggggattg agtttattgt 3540 atgccctaga acacaaggga gacagagcacttgtctccac tcaaggtgaa ttggcacatg 3600 cattgcggta tctagcttct gttgtctcccaaagctttat ggcttttggc gatattcttg 3660 aactacacaa gaagttcctg gagctctctggtggtattaa cagaattttt gagctcgatg 3720 agtttttgga tgcttctcag tcaggtgttacctcagaaaa tcaaacaagt cgtttggatt 3780 ctcaagatct actttccttt tcggaggtggatatcattac ccctgctcag aaattgatgg 3840 ctagcaagtt gtcgtgtgaa atagtttcagggaaaagcct gctcgtcaca ggtccaaatg 3900 gtagtggaaa gacttcagta tttagagtccttagagatat ctggcccact gtatgtggaa 3960 gacttaccaa accatcattg gatatcaaagaacttgggtc agggaatggc atgttttttg 4020 tcccgcagcg accttataca tgtttagggacactgagaga tcaaattata taccctctat 4080 ctaaagaaga agcagagaaa agggcagcaaagttgtacac cagtggagag agctcaacag 4140 aagctggaag cattctggat tctcatttgaaaaccattct ggagaatgtt cggttagttt 4200 atctcttgga aagagacgta ggtggttgggatgctactac caattgggaa gacatattat 4260 ctcttggaga gcaacagaga ttaggcatggcacgtttatt ctttcacagg ccgaagtttg 4320 gagtccttga tgaatgcaca aatgcgacgagtgttgatgt tgaggaacag ctctatagag 4380 ttgcacgaga catgggagtc actttcataacctcatcaca acggccggct ctgatcccat 4440 tccattcctt ggagctaagg ctgattgatggagaaggaaa ctgggagctc cgttcgatcg 4500 aacagacaac agagtgaact cagcaaaacatttttagaaa ggtctatata gttgttaaag 4560 aaaaaagtaa taaagttaaa gccattagacgatgcaagct atatggtatg tagtatatgg 4620 attcttcctc gatcgcaagg agtggaagagaatgcgtcga tgctagtgct tttgttagaa 4680 ttggaggatt tgatttgatt ctagatatatataaatgtag gcgattgaat tggtggagca 4740 ttttgagttc tcctatggag tatggtcttagctttgaaca aacaaagaat atagtgatca 4800 ctcaaataat gtacagttcg tttcaatttcctttgttggg attagttttt ctatcttata 4860 attaaaagaa tgaaattgaa gtgggcggccgcgaattcct gcagcccggg ggatccacta 4920 gttctagagc ggccgccacc gcggtggagctccagctttt gttcccttta gtgagggtta 4980 atttcgagct tggcgtaatc atggtcatagctgtttcctg tgtgaaattg tatccgctca 5040 caattccaca caacatacga gccggaagcataa 5073 2 1336 PRT Arabidopsis thaliana 2 Met Pro Ser Leu Gln Leu LeuGln Leu Thr Glu Arg Gly Arg Gly Leu 1 5 10 15 Val Ala Ser Arg Arg LysSer Ile Leu Leu Ala Ala Gly Ile Val Ala 20 25 30 Ala Gly Gly Thr Ala ValTyr Leu Lys Ser Arg Val Ala Ser Arg Arg 35 40 45 Pro Asp Ser Ser Arg LeuCys Asn Gly Gln Ser Asp Asp Asp Glu Thr 50 55 60 Leu Glu Lys Leu Thr AlaThr Asp Gln Asn Ala Lys Ile Thr Thr Lys 65 70 75 80 Lys Lys Lys Gly GlyGly Leu Lys Ser Leu Gln Val Leu Thr Ala Ile 85 90 95 Leu Leu Ser Gln MetGly Lys Met Gly Ala Arg Asp Leu Leu Ala Leu 100 105 110 Val Ala Thr ValVal Phe Arg Thr Ala Leu Ser Asn Arg Leu Ala Lys 115 120 125 Val Gln GlyPhe Leu Phe Arg Ala Ala Phe Leu Arg Arg Ala Pro Leu 130 135 140 Phe LeuArg Leu Ile Ser Glu Asn Ile Met Leu Cys Phe Met Leu Ser 145 150 155 160Thr Leu His Ser Thr Ser Lys Tyr Ile Thr Gly Ala Leu Ser Leu Arg 165 170175 Phe Arg Lys Ile Leu Thr Lys Ile Ile His Ser His Tyr Phe Glu Asn 180185 190 Met Val Tyr Tyr Lys Ile Ser His Val Asp Gly Arg Ile Thr His Pro195 200 205 Glu Gln Arg Ile Ala Ser Asp Val Pro Arg Phe Ser Ser Glu LeuSer 210 215 220 Asp Leu Ile Leu Asp Asp Leu Thr Ala Val Thr Asp Gly IleLeu Tyr 225 230 235 240 Ala Trp Arg Leu Cys Ser Tyr Ala Ser Pro Lys TyrIle Phe Trp Ile 245 250 255 Leu Ala Tyr Val Leu Gly Ala Gly Thr Ala IleArg Asn Phe Ser Pro 260 265 270 Ser Phe Gly Lys Leu Met Ser Lys Glu GlnGln Leu Glu Gly Glu Tyr 275 280 285 Arg Gln Leu His Ser Arg Leu Arg ThrHis Ser Glu Ser Ile Ala Phe 290 295 300 Tyr Gly Gly Glu Thr Arg Glu GluSer His Ile Gln Gln Lys Phe Lys 305 310 315 320 Asn Leu Val Ser His MetSer His Val Leu His Asp His Trp Trp Phe 325 330 335 Gly Met Ile Gln AspPhe Leu Leu Lys Tyr Leu Gly Ala Thr Val Ala 340 345 350 Val Ile Leu IleIle Glu Pro Phe Phe Ser Gly His Leu Arg Pro Asp 355 360 365 Asp Ser ThrLeu Gly Arg Ala Glu Met Leu Ser Asn Ile Arg Tyr His 370 375 380 Thr SerVal Ile Ile Ser Leu Phe Gln Ala Leu Gly Thr Leu Ser Ile 385 390 395 400Ser Ser Arg Arg Leu Asn Arg Leu Ser Gly Tyr Ala Asp Arg Ile His 405 410415 Glu Leu Met Ala Val Ser Arg Glu Leu Ser Gly Asp Asp Lys Ser Ser 420425 430 Phe Gln Arg Asn Arg Ser Arg Asn Tyr Leu Ser Glu Ala Asn Tyr Val435 440 445 Glu Phe Ser Asp Val Lys Val Val Thr Pro Thr Gly Asn Val LeuVal 450 455 460 Glu Asp Leu Thr Leu Arg Val Glu Gln Gly Ser Asn Leu LeuIle Thr 465 470 475 480 Gly Pro Asn Gly Ser Gly Lys Ser Ser Leu Phe ArgVal Leu Gly Gly 485 490 495 Leu Trp Pro Leu Val Ser Gly His Ile Val LysPro Gly Val Gly Ser 500 505 510 Asp Leu Asn Lys Glu Ile Phe Tyr Val ProGln Arg Pro Tyr Met Ala 515 520 525 Val Gly Thr Leu Arg Asp Gln Leu IleTyr Pro Leu Thr Ser Gly Gln 530 535 540 Glu Ser Glu Leu Leu Thr Glu IleGly Met Val Glu Leu Leu Lys Asn 545 550 555 560 Val Asp Leu Glu Tyr LeuLeu Asp Arg Tyr Gln Pro Glu Lys Glu Val 565 570 575 Asn Trp Gly Asp GluLeu Ser Leu Gly Glu Gln Gln Arg Leu Gly Met 580 585 590 Ala Arg Leu PheTyr His Lys Pro Lys Phe Ala Ile Leu Asp Glu Cys 595 600 605 Thr Ser AlaVal Thr Thr Asp Met Glu Glu Arg Phe Ala Ala Lys Val 610 615 620 Arg AlaMet Gly Thr Ser Cys Ile Thr Ile Ser His Arg Pro Ala Leu 625 630 635 640Val Ala Phe His Asp Val Val Leu Ser Leu Asp Gly Glu Gly Gly Trp 645 650655 Ser Val His Tyr Lys Arg Asp Asp Ser Ala Leu Leu Thr Asp Ala Glu 660665 670 Ile Asp Ser Val Lys Ser Ser Asp Thr Asp Arg Gln Asn Asp Ala Met675 680 685 Val Val Gln Arg Ala Phe Ala Ala Ala Arg Lys Glu Ser Ala ThrAsn 690 695 700 Ser Lys Ala Gln Ser Tyr Gln Thr Gln Leu Ile Ala Arg SerPro Val 705 710 715 720 Val Asp Lys Ser Val Val Leu Pro Arg Phe Pro GlnPro Gln Thr Ser 725 730 735 Gln Arg Ala Leu Pro Ser Arg Val Ala Ala MetLeu Asn Val Leu Ile 740 745 750 Pro Thr Ile Phe Asp Lys Gln Gly Ala GlnLeu Leu Ala Val Ala Cys 755 760 765 Leu Val Val Ser Arg Thr Leu Ile SerAsp Arg Ile Ala Ser Leu Asn 770 775 780 Gly Thr Thr Val Lys Tyr Val LeuGlu Gln Asp Lys Ala Ala Phe Val 785 790 795 800 Arg Leu Ile Gly Leu SerVal Leu Gln Ser Gly Ala Ser Ser Ile Ile 805 810 815 Ala Pro Ser Leu ArgHis Leu Thr Gln Arg Leu Ala Leu Gly Trp Arg 820 825 830 Ile Arg Leu ThrGln His Leu Leu Arg Asn Tyr Leu Arg Asn Asn Ala 835 840 845 Phe Tyr LysVal Phe His Met Ser Gly Asn Ser Ile Asp Ala Asp Gln 850 855 860 Arg LeuThr Arg Asp Leu Glu Lys Leu Thr Ala Asp Leu Ser Gly Leu 865 870 875 880Leu Thr Gly Met Val Lys Pro Ser Val Asp Ile Leu Trp Phe Thr Trp 885 890895 Arg Met Lys Leu Leu Thr Gly Gln Arg Gly Val Ala Ile Leu Tyr Thr 900905 910 Tyr Met Leu Leu Gly Leu Gly Phe Leu Arg Arg Val Ala Pro Asp Phe915 920 925 Gly Asp Leu Ala Gly Glu Glu Gln Gln Leu Glu Gly Lys Phe ArgPhe 930 935 940 Met His Glu Arg Leu Asn Thr His Ala Glu Ser Ile Ala PhePhe Gly 945 950 955 960 Gly Gly Ala Arg Glu Lys Ala Met Val Asp Lys LysPhe Arg Ala Leu 965 970 975 Leu Asp His Ser Leu Met Leu Leu Arg Lys LysTrp Leu Tyr Gly Ile 980 985 990 Leu Asp Asp Phe Val Thr Lys Gln Leu ProAsn Asn Val Thr Trp Gly 995 1000 1005 Leu Ser Leu Leu Tyr Ala Leu GluHis Lys Gly Asp Arg Ala Leu 1010 1015 1020 Val Ser Thr Gln Gly Glu LeuAla His Ala Leu Arg Tyr Leu Ala 1025 1030 1035 Ser Val Val Ser Gln SerPhe Met Ala Phe Gly Asp Ile Leu Glu 1040 1045 1050 Leu His Lys Lys PheLeu Glu Leu Ser Gly Gly Ile Asn Arg Ile 1055 1060 1065 Phe Glu Leu AspGlu Phe Leu Asp Ala Ser Gln Ser Gly Val Thr 1070 1075 1080 Ser Glu AsnGln Thr Ser Arg Leu Asp Ser Gln Asp Leu Leu Ser 1085 1090 1095 Phe SerGlu Val Asp Ile Ile Thr Pro Ala Gln Lys Met Ala Ser 1100 1105 1110 LysLeu Ser Cys Glu Ile Val Ser Gly Lys Ser Leu Leu Val Thr 1115 1120 1125Gly Pro Asn Gly Ser Gly Lys Thr Ser Val Phe Arg Val Leu Arg 1130 11351140 Asp Ile Trp Pro Thr Val Cys Gly Arg Leu Thr Lys Pro Ser Leu 11451150 1155 Asp Ile Lys Glu Leu Gly Ser Gly Asn Gly Met Phe Phe Val Pro1160 1165 1170 Gln Arg Pro Tyr Thr Cys Leu Gly Thr Leu Arg Asp Gln IleIle 1175 1180 1185 Tyr Pro Leu Ser Lys Glu Glu Ala Glu Lys Arg Ala AlaLys Leu 1190 1195 1200 Tyr Thr Ser Gly Glu Ser Ser Thr Glu Ala Gly SerIle Leu Asp 1205 1210 1215 Ser His Leu Lys Thr Ile Leu Glu Asn Val ArgLeu Val Tyr Leu 1220 1225 1230 Leu Glu Arg Asp Val Gly Gly Trp Asp AlaThr Thr Asn Trp Glu 1235 1240 1245 Asp Ile Leu Ser Leu Gly Glu Gln GlnArg Leu Gly Met Ala Arg 1250 1255 1260 Leu Phe Phe His Arg Pro Lys PheGly Val Leu Asp Glu Cys Thr 1265 1270 1275 Asn Ala Thr Ser Val Asp ValGlu Glu Gln Leu Tyr Arg Val Ala 1280 1285 1290 Arg Asp Met Gly Val ThrPhe Ile Thr Ser Ser Gln Arg Pro Ala 1295 1300 1305 Leu Ile Pro Phe HisSer Leu Glu Leu Arg Leu Ile Asp Gly Glu 1310 1315 1320 Gly Asn Trp GluLeu Arg Ser Ile Glu Gln Thr Thr Glu 1325 1330 1335 3 29 DNA Arabidopsisthaliana 3 ctctctctct atctctatct ctcgatttg 29 4 29 DNA Arabidopsisthaliana 4 tctagctaag tggttgttgt tgttgttac 29 5 29 DNA Arabidopsisthaliana 5 gccgttgaga ttcacatggt ctagaattc 29 6 29 DNA Arabidopsisthaliana 6 atgaaacggt ggctagagtt acctgagta 29 7 29 DNA Arabidopsisthaliana 7 tacactttta tctacaacag gtgatcttg 29 8 29 DNA Arabidopsisthaliana 8 gcaattatag aagatgcacc actttggag 29 9 29 DNA Arabidopsisthaliana 9 catagaatgc tatgctttcc gaatgagtc 29 10 29 DNA Arabidopsisthaliana 10 cccaatgaga tctttagtgt ctctagcca 29 11 29 DNA Arabidopsisthaliana 11 cccaatgaga tctttagtgt ctctagcca 29

1. An isolated nucleic acid comprising a nucleotide sequence whichencodes a polypeptide which functions as a fatty acid transporter inplants selected from the group consisting of: (i) a nucleic acidsequence depicted in SEQ ID NO: 1, (ii) a nucleic acid sequence which isderived from the sequence depicted in SEQ ID NO: 1 according to thedegeneracy of the genetic code, (iii) derivatives of the sequencedepicted in SEQ ID NO: 1, which encodes polypeptides having at least 30%homology to the sequence encoding amino acid sequences depicted in SEQID NO: 2 and which sequences function as a fatty acid transporter.
 2. Anisolated nucleic acid according to claim 1 comprising a nucleotidesequence which hybridizes to, or is at least about 50%, homologous tothe nucleotide sequence in SEQ ID NO: 1, or a portion thereof.
 3. Anisolated nucleic acid molecule according to claim 2 which is at leastabout 60%, 70%, 80% or 90%, homologous to the nucleotide sequence in SEQID NO: 1, or a portion thereof.
 4. An isolated nucleic acid moleculeaccording to claim 2 which is at least about 95%, 96%, 97%, 98%, 99% ormore homologous to the nucleotide sequence in SEQ ID NO: 1, or a portionthereof.
 5. The isolated nucleic acid molecule according to claim 1which encodes a polypeptide which functions as a transporter of acylCoAs of varying chain lengths, degree of unsaturation and substitution,and their analogues and derivatives and/or transporter of amphipathicmolecules, 2,4 dichlorophenoxybutyric acid and/or indole butyric acidand their analogues and derivatives.
 6. The isolated nucleic acidaccording to claim 1 which corresponds to a naturally-occurring nucleicacid molecule.
 7. The isolated nucleic acid according to claim 1 whichencodes a naturally-occurring Arabidopsis thaliana fatty acidtransporter, or a biologically active portion thereof.
 8. A geneconstruct comprising an isolated nucleic acid having the sequence SEQ IDNO: 1 as claimed in claim 1 wherein the nucleic acid is functionallylinked to one or more regulatory signals.
 9. A The gene constructaccording to claim 8 wherein the regulatory signal comprises a sequenceencoding a promoter.
 10. A The gene construct according to claim 8,further comprising an additional gene associated with metabolism,catabolism or synthesis of an endogenous or exogenous product.
 11. Avector comprising the nucleic acid of claim
 1. 12. A host cellcomprising the vector according to claim
 11. 13. Use of the nucleic acidof claim 1 and/or the protein or polypeptide encoded thereby in any oneor more of the following processes: regulating fatty acid or acyl CoAtransport across peroxisome and/or glyoxisome and/or cellular membranes;regulating growth; regulating seed development and; modulating fattyacid utilisation by a plant.
 14. A method of regulating any one or moreof the following processes: fatty acid transport across peroxisomeand/or glyoxisome membranes; seed development and; fatty acidutilisation by a plant comprising genetically engineering a plant cellor tissue or seed so as to enhance or reduce/prevent expression of thenucleic acid according to claim
 1. 15. A method of modifying a plantcell to increase or decrease the transport of some or all fatty acidsacross cell membranes and/or to increase or prevent their breakdowncomprising mutating, disabling or deleting the nucleic acid according toclaim 1 or altering its expression levels in specific tissues or atspecific times or increasing the number of copies of the nucleic acid ascompared to the wild-type.
 16. A transgenic plant comprising anincreased or decreased number of gene copies of a nucleic acid moleculeaccording to claim
 1. 17. The transgenic plant according to claim 16wherein the plant has increased or decreased expression of an activefatty acid transporter protein.
 18. A method of regulating fatty acidlevels in plants comprising genetically engineering a plant cell ortissue or seed so as to disrupt, deactivate, disable, mutate, delete,knockout or render transcriptionally ineffective the nucleic acidaccording to claim
 1. 19. A plant cell and/or a plant tissue and/orplant and/or plant seed that has been modified so that it does notcontain a transcriptionally activated/activatable form of the nucleicacid molecule according to claim 1 or contains a reduced number ofcopies of the nucleic acid as compared to the wild-type.
 20. A plantgenerated from a plant cell and/or plant tissue and/or plant and/orplant seed which contains a disrupted, deactivated, disabled, mutated,deleted, knocked-out or rendered transcriptionally ineffective nucleicacid according to claim 1 as compared to the wild-type.
 21. A plant celland/or a plant tissue and/or plant and/or plant seed comprising: (i) anincreased number of copies of the nucleic acid according to claim 1 ascompared to the wild-type; (ii) increased transcription of the nucleicacid according to claim 1 as compared to the wild-type; or (iii) adiffering number of copies of the nucleic acid according to claim 1depending on the time of development of the plant.
 22. A plant generatedfrom a plant cell and/or plant tissue and/or plant and/or plant seedcomprising: (i) an increased number of copies of the nucleic acidaccording to claim 1 as compared to the wild-type; (ii) increasedtranscription of the nucleic acid according to claim 1 as compared tothe wild-type; or (iii) a differing number of copies of the nucleic acidaccording to claim 1 depending on the time of development of the plant.23. A primer comprising the nucleotide sequence of any one of SEQ IDNOS: 3 to 10 or parts thereof capable of recognising the nucleic acidaccording to claim
 1. 24. Use of a primer comprising the nucleotidesequence of any one of SEQ ID NOS: 3 to 10 or parts thereof capable ofrecognising the nucleic acid according to claim
 1. 25. A method ofidentifying plant material selected from a plant cell and/or planttissue and/or plant and/or plant seed comprising a disrupted,deactivated, disabled, mutated, deleted, knocked-out or renderedtranscriptionally ineffective nucleic acid according to claim 1comprising contacting the plant material with a primer comprising thenucleotide sequence of any one of SEQ ID NOS: 3 to
 10. 26. A polypeptideor protein comprising the amino acid sequence of SEQ ID NO: 2 or a partor homologue or derivative thereof which functions as an integralmembrane protein and is capable of transporting molecules across amembrane.
 27. The polypeptide or protein according to claim 26 that isan ATP-binding cassette transporter protein.
 28. The polypeptide orprotein according to claim 26 that is a peroxisomal ABC transporter. 29.A vector comprising the gene construct of claim
 8. 30. A host cellcomprising the gene construct according to claim
 29. 31. Use of the geneconstruct of claim 8 in any one or more of the following processes:regulating fatty acid or acyl CoA transport across peroxisome and/orglyoxisome and/or cellular membranes; regulating growth; regulating seeddevelopment and; modulating fatty acid utilisation by a plant.
 32. Useof the vector of claim 11 in any one or more of the following processes:regulating fatty acid or acyl CoA transport across peroxisome and/orglyoxisome and/or cellular membranes; regulating growth; regulating seeddevelopment and; modulating fatty acid utilisation by a plant.
 33. Useof the host cell of claim 12 in any one or more of the followingprocesses: regulating fatty acid or acyl CoA transport across peroxisomeand/or glyoxisome and/or cellular membranes; regulating growth;regulating seed development and; modulating fatty acid utilisation by aplant.